Abstract Silicosis is the most prevalent and fatal occupational disease with no effective therapeutics, and currently used drugs cannot reverse the disease progress. Worse still, there are still challenges to be addressed to fully decipher the intricated pathogenesis. Thus, specifying the essential mechanisms and targets in silicosis progression then exploring anti-silicosis pharmacuticals are desperately needed. In this work, multi-omics atlas was constructed to depict the pivotal abnormalities of silicosis and develop targeted agents. By utilizing an unbiased and time-resolved analysis of the transcriptome, proteome and phosphoproteome of a silicosis mouse model, we have verified the significant differences in transcript, protein, kinase activity and signaling pathway level during silicosis progression, in which the importance of essential biological processes such as macrophage activation, chemotaxis, immune cell recruitment and chronic inflammation were emphasized. Notably, the phosphorylation of EGFR (p-EGFR) and SYK (p-SYK) were identified as potential therapeutic targets in the progression of silicosis. To inhibit and validate these targets, we tested fostamatinib (targeting SYK) and Gefitinib (targeting EGFR), and both drugs effectively ameliorated pulmonary dysfunction and inhibited the progression of inflammation and fibrosis. Overall, our drug discovery with multi-omics approach provides novel and viable therapeutic strategies for the treatment of silicosis.
Pulmonary hypertension (PH) is a fatal disorder characterized by pulmonary vascular remodeling and obstruction. The phosphodiesterase 4 (PDE4) family hydrolyzes cyclic AMP (cAMP) and is comprised of four subtypes (PDE4A–D). Previous studies have shown the beneficial effects of pan-PDE4 inhibitors in rodent PH; however, this class of drugs is associated with side effects owing to the broad inhibition of all four PDE4 isozymes. Here, we demonstrate that PDE4B is the predominant PDE isozyme in lungs and that it was upregulated in rodent and human PH lung tissues. We also confirmed that PDE4B is mainly expressed in the lung endothelial cells (ECs). Evaluation of PH in Pde4b wild type and knockout mice confirmed that Pde4b is important for the vascular remodeling associated with PH. In vivo EC lineage tracing demonstrated that Pde4b induces PH development by driving endothelial-to-mesenchymal transition (EndMT), and mechanistic studies showed that Pde4b regulates EndMT by antagonizing the cAMP-dependent PKA-CREB-BMPRII axis. Finally, treating PH rats with a PDE4B-specific inhibitor validated that PDE4B inhibition has a significant pharmacological effect in the alleviation of PH. Collectively, our findings indicate a critical role for PDE4B in EndMT and PH, prompting further studies of PDE4B-specific inhibitors as a therapeutic strategy for PH.
Background Pulmonary hypertension (PH) is a life-threatening disease featuring pulmonary vessel remodelling and perivascular inflammation. The effect, if any, of eosinophils (EOS) on the development of PH remains unclear. Methods EOS infiltration and chemotaxis were investigated in peripheral blood and lung tissues from pulmonary arterial hypertension (PAH) patients without allergic history and from sugen/hypoxia-induced PH mice. The role of EOS deficiency in PH development was investigated using GATA1- deletion (ΔdblGATA) mice and anti-interleukin 5 antibody-treated mice and rats. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was conducted to identify the critical oxylipin molecule(s) produced by EOS. Culture supernatants and lysates of EOS were collected to explore the mechanisms in co-culture cell experiments. Results There was a lower percentage of EOS in peripheral blood but higher infiltration in lung tissues from PAH patients and PH mice. PAH/PH lungs showed increased EOS-related chemokine expression, mainly C-C motif chemokine ligand 11 derived from adventitial fibroblasts. EOS deficiency aggravated PH in rodents, accompanied by increased neutrophil and monocyte/macrophage infiltration. EOS highly expressed arachidonate 15-lipoxygenase (ALOX15). 14-hydroxy docosahexaenoic acid (14-HDHA) and 17-HDHA were critical downstream oxylipins produced by EOS, which showed anti-inflammatory effects on recruitment of neutrophils and monocytes/macrophages through N-formyl peptide receptor 2. They also repressed pulmonary artery smooth muscle cell (PASMC) proliferation by activating peroxisome proliferator-activated receptor γ and blunting Stat3 phosphorylation. Conclusions In PH development without external stimuli, peripheral blood exhibits a low EOS level. EOS play a protective role by suppressing perivascular inflammation and maintaining PASMC homeostasis via 14/17-HDHA.
Abstract Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by pathological vascular remodeling in the lung. Immunoglobulin E (IgE) is known to participate in aortic vascular remodeling, but whether IgE mediates pulmonary vascular remodeling in PH is unknown. Here, we found serum IgE elevation in PAH patients, hypoxia-induced PH mice and monocrotaline (MCT)-induced PH rats. Combining animal model of PH with single-cell RNA sequencing, we found IgE production in the lung tissues of PH mice. Neutralizing IgE with an anti-IgE antibody was effective in preventing PH development in mice and rat models. The IgE receptor FcεRIα was also upregulated in PH lung tissues and Fcer1a deficiency prevented the development of PH in mice. Single-cell RNA-seq revealed that FcεRIα was mostly expressed in mast cells, and mast cell-specific Fcer1a knockout protected against PH in mice. Further mechanistic experiments revealed that IgE-activated mast cells produced interleukins IL6 and IL13, which subsequently promoted vascular muscularization. Clinically approved IgE antibody Omalizumab alleviated the progression of established PH in rats. Using genetic and pharmacological approaches, we have demonstrated that blocking IgE- FcεRIα signaling may hold potential for the treatment of PAH.
Inoculation of mice with the murine NFSA cell line caused the formation of large tumors with necrotic tumor cores. FACS analysis revealed accumulations of CD11b(+) cells in the tumors. Microarray analysis indicated that the NFSA cells expressed a high level of the pro-inflammatory factor interleukin-18 (il-18), which is known to play a critical role in macrophages. However, little is known about the physiological function of IL-18-stimulated macrophages. Here, we provide direct evidence that IL-18 enhances the phagocytosis of RAW264 cells and peritoneal macrophages, accompanied by the increased expression of tumor necrosis factor (tnf-α), interleukin-6 (il-6) and inducible nitric oxide synthase (Nos2). IL-18-stimulated RAW264 cells showed an enhanced cytotoxicity to endothelial F-2 cells via direct cell-to-cell interaction and the secretion of soluble mediators. Taken together, our results demonstrate that tumor-derived IL-18 plays an important role in the phagocytosis of macrophages and that IL-18-stimulated macrophages may damage tumor endothelial cells.
Pulmonary arterial hypertension (PAH) is a common and fatal complication of systemic lupus erythematosus (SLE). Whether the BMP receptor deficiency found in the genetic form of PAH is also involved in SLE-PAH patients remains to be identified. In this study, we employed patient-derived samples from SLE-associated PAH (SLE-PAH) and established comparable mouse models to clarify the role of BMP signaling in the pathobiology of SLE-PAH. Firstly, serum levels of LPS and autoantibodies (auto-Abs) directed at BMP receptors were significantly increased in patients with SLE-PAH compared with control subjects, measured by ELISA. Mass cytometry was applied to compare peripheral blood leukocyte phenotype in patients prior to and after treatment with steroids, which demonstrated inflammatory cells alteration in SLE-PAH. Furthermore, BMPR2 signaling and pyroptotic factors were examined in human pulmonary arterial endothelial cells (PAECs) in response to LPS stimulation. Interleukin-8 (IL-8) and E-selectin (SELE) expressions were up-regulated in autologous BMPR2+/R899X endothelial cells and siBMPR2-interfered PAECs. A SLE-PH model was established in mice induced with pristane and hypoxia. Moreover, the combination of endothelial specific BMPR2 knockout in SLE mice exacerbated pulmonary hypertension. Pyroptotic factors including gasdermin D (GSDMD) were elevated in the lungs of SLE-PH mice, and the pyroptotic effects of serum samples isolated from SLE-PAH patients on PAECs were analyzed. BMPR2 signaling upregulator (BUR1) showed anti-pyroptotic effects in SLE-PH mice and PAECs. Our results implied that deficiencies of BMPR2 signaling and proinflammatory factors together contribute to the development of PAH in SLE.
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37 Background: Accumulating evidence indicates endothelial progenitor cells (EPCs) play a major role in regulating pulmonary vascular remodeling during pulmonary arterial hypertension (PAH) development. However, there is no reliable method of real-time trafficking and quantification of transferred EPCs. We aimed to detect the homing of EPCs in health and PAH rats by 89Zr-oxine labeling PET imaging. Methods: EPCs, isolated from human peripheral blood, was identified by specific EPCs biomarkers. The effect of 89Zr-oxine labeling on EPCs cell viability and proliferation was evaluated in vitro. 89Zr-labeled EPC cells (2 × 106 cells, 90 kBq/106 cells) were transferred intravenously to health and PAH rats and serial microPET/CT images were obtained. Results: EPCs were characterized and efficiently labeled. In vitro viability and proliferation were not significantly reduced when labeled with 90 kBq per million cells. Intravenously administered 89Zr-labeled EPCs distributed primarily to the lung at 1 h and then subsequently migrated to the liver and spleen. The liver and spleen showed moderate accumulation with the highest %ID/g value of 5.30 ± 1.10 and 2.95 ± 0.54 at 72 h, respectively. The kidney and heart were also slightly visualized. Furthermore, microPET/CT results showed significantly higher accumulation of EPCs in lung of PAH than control group. Conclusions: 89Zr-oxine can be used to delineate EPCs in lung of PAH by PET imaging and may provide a noninvasive EPCs monitoring tool.
Figure legendA. Immunostaining and flow cytometric analysis of EPCs revealed expression of endothelial cell-specific markers CD31 (green), CD144 (green), and vWF (green), CD146 (red), KDR and the progenitor cell marker CD34. Nuclei were counterstained with DAPI (blue). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; vWF, von Willebrand factor; KDR, kinase insert domain receptor. B. Whole-body microPET/CT imaging of 89Zr-oxine EPCs in health rats. Maximum intensity projections (MIPs) of representative mice are shown at several time points after injection. C. Axial section showing the microPET/CT signal of 89Zr-oxine EPCs at the lung of the representative health and pulmonary arterial hypertension (PAH) rats. D. Quantification of PET images in the lung of health and PAH rats treated with 89Zr-oxine EPC cells over 10 days.
Acknowledgments:This work was sponsored in part by the National Natural Science Foundation of China (Grant No. 81571713), Capital’s Funds for Health Improvement and Research(CFH) (Grant No. 2016-2-40115), CAMS Innovation Fund for Medical Sciences(CIFMS)(Grant No. 2016-I2M-4-003, 2017-I2M-3-001, 2018-I2M-3-001). No other potential conflicts of interest relevant to this article exist.
Endothelial progenitor cells (EPCs) play a major role in regulating pulmonary vascular remodeling during pulmonary arterial hypertension (PAH) development. Several preclinical and clinical trials of EPCs transplantation have been performed for the treatment of PAH. However, there is no reliable method to monitor real-time cell trafficking and quantify transplanted EPCs. Here in this paper we isolated EPCs from human peripheral blood, identified their functional integrity, and efficiently labeled the EPCs with 89Zr-oxine and DiO. Labeled EPCs were injected into the tail vein of normal and PAH rats to be tracked in vivo. From the microPET/CT images, we found EPCs were distributed primarily in the lung at 1 h and then migrated to the liver and spleen. We could observe the 3,3' dioctadecyloxacarbocyanine perchlorate (DiO)-labeled EPCs binding in the pulmonary vasculature by CellVizio confocal. The result of quantitative analysis revealed significantly higher accumulation of EPCs in the lungs of PAH rats than in those of healthy rats. The distribution and higher accumulation of EPCs in the lungs of PAH rats could help to evaluate the safety and provide evidence of effectiveness of EPC therapy.
We previously demonstrated that IL ‐18 and CCL 11 were highly expressed in an NFSA tumor cell line that showed limited angiogenesis and severe necrosis. However, IL ‐18 was not responsible for the immune cell accumulation and necrosis. Here, we attempted to clarify the relevance of CCL 11 in angiogenesis and tumor formation. We established CCL 11‐overexpressing MS ‐K cell clones ( MS ‐K‐ CCL 11) to assess the role of CCL 11 in immune cell accumulation and angiogenesis. The MS ‐K‐ CCL 11 cells did not form tumors in mice. MS ‐K‐ CCL 11‐conditioned medium ( CM ) and recombinant CCL 11 induced macrophage and eosinophil differentiation from bone marrow cells. The MS ‐K‐ CCL 11‐ CM effectively recruited the differentiated eosinophils. Furthermore, the eosinophils damaged the MS ‐K, NFSA and endothelial cells in a dose‐dependent manner. Administration of an antagonist of CCR 3, a CCL 11 receptor, to NFSA tumor‐bearing mice restored the blood vessel formation and blocked the eosinophil infiltration into the NFSA tumors. Furthermore, other CCL 11‐overexpressing LM 8 clones were established, and their tumor formation ability was reduced compared to the parental LM 8 cells, accompanied by increased eosinophil infiltration, blockade of angiogenesis and necrosis. These results indicate that CCL 11 was responsible for the limited angiogenesis and necrosis by inducing and attracting eosinophils in the tumors.
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